Ligand interactions with membrane proteins are responsible for a multitude of cell adhesion, signaling, and regulatory events. This diversity of function makes membrane proteins important drug targets. G-protein coupled receptors (GPCRs) are one family of membrane proteins that comprise nearly half of existing drug targets (Drews (1996), Nat. Biotechnol., 11:1516-1518, Stadel, et al. (1997), Trends Pharmacol. Sci., 18:430-437, Wise, et al. (2002), Drug Discovery Today, 7:235-246). Another 5% of drug targets are comprised of membrane-embedded ion channels, and many of the remaining targets are also membrane proteins. These topologically complex membrane proteins span the lipid bilayer of the cell multiple times and usually serve as receptors to mediate communication between a cell's function and its exterior environment. Drugs targeting membrane proteins include antipsychotics, antihistamines, beta-blockers, anti-migraine drugs, anti-ulcer drugs, and analgesics.
Despite their importance, proteins that span the membrane multiple times present a unique set of challenges for ligand binding and drug discovery studies because they require a lipid environment to maintain native structure. For example, GPCRs weave in and out of the cell membrane seven times, so cannot be extracted from the lipid bilayer without disrupting their structure. While detergent conditions can occasionally be found that allow native structure to be maintained in solution, this is an empirical and very time-consuming process, and even then stability is only transient.
As a result, interaction studies involving membrane proteins typically use whole cells or vesicles derived from cell membranes, where the protein of interest is but a minor component, making both adequate sensitivity and specificity more difficult to achieve. Living cells are cumbersome to grow, must be maintained in a high protein (serum) environment, and present a moving target as receptors are internalized, altered by intracellular events, and recycled. Cells also have severe limitations in their application to biosensors and other microfluidic devices, most notably in their size, sensitivity to environmental conditions, and heterogeneous cell surface. Membrane vesicles, prepared by mechanically disrupting cells, are a common source of membrane protein for many drug screens currently conducted. However, membrane vesicles are heterogeneous, impure, and not particularly stable. The receptors within them may be misoriented, a minor component of total protein, and derived from intracellular organelles.
Thus, there remains a need for the development of a broad methodology that permits the rapid purification of a wide spectrum of membrane and cellular proteins. The present invention satisfies this need. Further, there is a need for assays that permit the study of membrane protein interactions, and the present invention also satisfies this need. Further, there is a need for vesicles that facilitate the elicitation of an immune response against membrane proteins to be mounted, particularly for the generation of monoclonal antibodies, humoral response, cellular response, and vaccines, and the present invention also satisfies this need. The present invention fulfills these needs as well others.
Human pathogens enter their host cells, and eventually kill or weaken them, using cellular receptors. In nearly all cases, these cellular receptors are membrane proteins on the cell surface. Identifying these membrane proteins and linking infectious agents to their receptors offers direct insight into disease pathogenesis. For example, HIV uses a fusion co-receptor, typically either the chemokine receptors CXCR4 or CCR5 (Doranz (2000), Emerging Therapeutic Targets, 4:423-437). Strains of HIV that use the co-receptor CXCR4 are correlated with increased disease pathogenicity, while strains that use the co-receptor CCR5 are responsible for transmission of the virus. Preventing receptor binding is a common strategy for treating or preventing pathogen infection.
Cellular receptors are not only involved in pathogen binding, fusion, and entry, but are also involved with other pathogenic processes. For example, many pathogens encode toxins that facilitate virulence of the pathogen. Examples include anthrax toxin (Bradley, et al. (2003), Biochem Pharmacol, 65:309-314), HHV8-encoded chemokines (vMIP-2) (Holst, et al. (2003), Contrib Microbiol, 10:232-252), cytomegalovirus chemokine UL146 (Penfold, et al. (1999), Proc. Natl. Acad. Sci. USA, 96:9839-9844), and pox virus-encoded chemokine inhibitors (vCCI, M-T1, M-T7) (Lalani, et al. (1997), J. Leukoc. Biol., 62:570-576, Smith, et al. (1997), Virology, 236:316-327). In many cases, identifying and inhibiting these toxins and their receptors can explain the lethal effects of the pathogen and neutralize their actions.
The techniques currently used to identify pathogen receptor interactions are generally slow and laborious. In many cases, such techniques require transfection of individual receptors, the growth of cells, and the use of the pathogen in its infectious form. In other cases, protein interactions are detected using immunoprecipitation, Western blotting, or radiolabeled proteins. For example, the HIV co-receptors CCR5 and CXCR4 were identified over ten years after HIV and its primary receptor, CD4, were discovered.
Optical biosensors are a class of instruments that can detect affinities of intramolecular interaction from picomolar to micromolar concentrations, in real-time, and without labels. Biosensors can also yield pharmacological information (kinetic and equilibrium binding constants) that other assays cannot (see (Canziani, et al. (1999), Methods, 19:253-269, Day, et al. (2002), Protein Science, 11:1017-1025) for review). Biosensors have been integrated into both drug discovery and diagnostics.
The most widely used optical biosensor, the BIACORE™, is based on surface plasmon resonance (SPR), which measures changes in refractive index at the sensor surface. The BIACORE platform consists of a flow cell with three inert walls (sides and floor) and a gold ceiling that is chemically modified to attach biomolecules. During usage, binding of protein in solution to tethered protein on the biosensor surface is monitored by changes in refractive index at the chip surface. A number of new biosensor devices are emerging that operate on similar principles.
Biosensors can also operate by measuring changes in spectroscopic measurements, such as reflectance, absorbance, transmission, or resonance. For example, microcantilever-based biosensors operate by detecting mechanical deflections of light reflecting from microcantilevers. The microcantilevers can be conjugated with an antibody, a protein, a ligand, a small molecule, a peptide, or a lipoparticle. The binding partner that deflects the cantilever can also be an antibody, a protein, a ligand, a small molecule, a peptide, or a lipoparticle.
Other biosensors are based on surface plasmon resonance and operate using an array format (on the world wide web at htsbiosystems.com). Other biosensors are based on colorimetric diffraction grating and operate using an array format (Cunningham, et al. (2002), Sensors and Actuators, B81:316-328, Cunningham, et al. (2002), Sensors and Actuators, B85:219-226, Lin, et al. (2002), Biosens Bioelectron, 17:827-834). Other biosensors are based on acoustic resonators (Cooper, et al. (2001), Nat Biotechnol, 19:833-7).
Such biosensors, however, have not been widely suitable for membrane proteins because: 1) biosensor detection signals are a function of distance from the surface and structures larger than 200 nm yield poor signals (e.g. cells, membrane vesicles), 2) the purity of molecules tethered to the biosensor surface is directly proportional to the signal-to-noise ratio, 3) the use of microfluidic channels limits the size of flow components, 4) the nature of biosensor detection restricts nearly all such devices to soluble molecules, and 5) removal of membrane proteins from their native lipid environment destroys their structure.
Only a handful of proof-of-concept studies have detected binding to membrane proteins using optical biosensors, but not using lipoparticles (Bieri, et al. (1999), Nat. Biotechnol., 17:1105-1108, Cooper, et al. (2000), Analytical Biochemistry, 277:196-205, Cooper, et al. (1998), Biochim Biophys Acta, 1373: Heyes, et al. (1998), Biochemistry, 37:507-522, Karlsson, et al. (2002), Analytical Biochemistry, 300:132-138, Salamon, et al. (2000), Biophysical Journal, 79:2463-2474, Salamon, et al. (1994), Biochemistry, 33:13706-13711, Salamon, et al. (1996), Biophysical Journal, 71:283-294). All of these studies used detergent-solubilized membrane proteins, and most focused on one prototype protein where solubilization conditions have been well studied (rhodopsin). Detergent-solubilized membrane proteins have been used to investigate membrane protein interactions, although this approach has significant drawbacks. As discussed previously, G-protein coupled receptors (GPCRs) weave in and out of the cell membrane seven times, and thus cannot be extracted from the lipid bilayer without disrupting their structure.
Doms et al. (U.S. Patent Publication 2002/0183247 A1) discusses using biosensors to detect interactions between membrane proteins on lipoparticles and their binding partners, however improvements are still needed.
Thus, there remains a need for improved methods for using membrane proteins with optical biosensors. One aspect of the present invention is to use arrays of membrane proteins in conjunction with optical biosensors to recreate the cell surface in vitro. This will result in a product that will consist of a biosensor chip containing the thousands of membrane proteins encoded by the human genome. There is also a need for improved methods and techniques to identify receptors for viral entry. There is also a need for improved methods of identifying receptors for ligands or drugs. There is also a need for improved methods of identifying unknown pathogens or substances in a sample. The present invention satisfies these needs and others.
Biological probes comprise one or both of two basic functional features: a targeting component and a reporting component. The targeting component interacts with the structure or molecule of interest and defines the ability of the probe to discriminate target structures or events. The reporting component signals target interaction and defines detection parameters such as sensitivity. Most biological probes in current use (e.g. antibodies, fluorescent proteins, ion-sensitive dyes) are single molecule structures, and thus usually possess a single reporting domain and either no or one targeting domain.
Molecules designed to detect the presence of specific biological targets or report the occurrence of biological events are known broadly as molecular probes. Traditional imaging probes comprise a recognition component (in this context defined as a “targeting” domain) which binds to a target molecule, and a signaling component (in this context defined as a “reporter”) which illuminates it (reviewed in (Massoud, et al. (2003), Genes Dev, 17:545-80, Molecular Probes Handbook (2003), Zhang, et al. (2002), Nat Rev Mol Cell Biol, 3:906-18)). The most widely used reporters emit an electromagnetic signal in the visual spectrum (bioluminescence or fluorescence), but radioactive and magnetic signals are also of medical importance for imaging techniques such as autoradiography, positron emission tomography (PET), and magnetic resonance imaging (MRI). Examples of visual reporters include fluorescent proteins, luminescent substrates, quantum dots, and fluorescent dyes. Biological probes incorporating these types of reporters are used to physically map cell structures and tissue architecture, as well as to monitor (and correlate) biological functions. The cellular structures and events that are emerging as important targets of molecular probes include subcellular components involved in gene transcription, cell growth and proliferation, cell migration and second messenger pathways.
With the increasing need to monitor smaller targets, and to dissect complex cellular functions at greater resolution, has arisen a requirement for the development of more sophisticated probes with improved localization and reporting characteristics. Cells are able to interpret the meaning of individual signaling events, for example, because each is part of a larger cellular response which includes sequential second messenger activity and spatial localization of signaling components. In contrast, most of the probes used in research and diagnostics to interpret such cell events rely on simple end-point measurements of a single event, target, or phenomenon (e.g. fluctuations in cytosolic calcium), and often fail to resolve the target to a sub-cellular location. The two most important characteristics of emerging biomedical imaging strategies are the ability to localize and align structural and functional information at tissue, cellular and sub-cellular levels, and the ability to exploit ‘multimodal’ detection systems (more than one reporter being simultaneously detected or correlated). Improvements in these properties can allow superior spatial localization of abnormalities in vivo, as well as structure-function correlation on the subcellular level (Massoud, et al. (2003), Genes Dev, 17:545-80). These ‘new generation’ probes are playing an increasingly important role in defining molecular events in the fields of cancer biology, cell biology, and gene therapy, for example in the detection of tumor markers and in tracking the delivery and function of gene therapy vectors (Jendelova, et al. (2004), J Neurosci Res, 76:232-43, Ray, et al. (2003), Cancer Res, 63:1160-5, Townsend, et al. (2001), Eur Radiol, 11:1968-74, Wu, et al. (2003), Nat Biotechnol, 21:41-6). Most existing single-molecule probes possess inherent limitations in these properties due to difficulties in integrating complex targeting and reporting systems. Although multiple probes can be introduced to simultaneously detect several events, most reporters cannot be targeted to desired cellular or subcellular structures to more accurately differentiate signaling events. Those probes that can be localized (e.g. fluorescent antibodies) usually contain only a small number (1-4) of reporter fluorophores per molecule, limiting signal amplification and detection.
A major obstacle in constructing improved imaging probes is not simply the development of new reporter molecules, but the development of a suitably sophisticated format in which complex and multiple reporters can be linked, and, importantly, controlled for target localization. Probes that can be spatially localized at the nanometer scale, that can amplify infrequent signals, and that can compartmentalize multiple reporters simultaneously, could have a major impact on developing subcellular and nano-scale applications in biomedical research and diagnostics.
A variety of foreign soluble proteins can also be incorporated into retroviruses. The incorporation of green fluorescent protein (GFP) into retroviruses has been used in a number of studies to understand aspects of the retroviral lifecycle such as budding, assembly, and infection (Andrawiss, et al. (2003), J Virol, 77:11651-60, Dalton, et al. (2001), Virology, 279:414-421, McDonald, et al. (2002), J. Cell Biol., 159: McDonald, et al. (2003), Science, 300:1295-7). In addition, labeling of viruses with fluorescent reporters has been used on several occasions to understand the early stages of virus fusion, endocytosis, and nuclear migration (Bartlett, et al. (1998), Nat Med, 4:635-7, Leopold, et al. (2000), Hum Gene Ther, 11:151-65, McDonald, et al. (2002), J. Cell Biol., 159: McDonald, et al. (2003), Science, 300:1295-7). An additional study demonstrated proof-of-concept incorporation of a different foreign protein fused to Gag (cytochrome c from yeast) (Weldon, et al. (1990), J Virol, 64:4169-79, Wills (1989), Nature, 340:323-4; U.S. Pat. No. 5,175,099). Probes attached to viruses have been limited to antibodies or fluorescent proteins that typically have limited life-spans and have never reported anything more than the location of the virus. Similar work has also been performed to study the phagocytosis of fluorescent yeast and bacteria (a product currently sold by Molecular Probes as ‘BioParticles’) (Giaimis, et al. (1994), Cytometry, 17:173-8, Haugland (2003), Oben, et al. (1988), J Immunol Methods, 112:99-103, Perticarari, et al. (1994), J Immunol Methods, 170:117-24, Ragsdale, et al. (1989), J Immunol Methods, 123:259-67), but virus-based bioparticles have never been developed. The incorporation of proteins of desired specificity and function into retroviruses has never been pursued for probe purposes.
There has been a desire and long-felt need for vehicles that can encapsulate and target multimodal probes to be used in the imaging field (Massoud, et al. (2003), Genes Dev, 17:545-80). The use of liposomes and related lipid structures is one approach that others have pursued. For example, synthetic beads conjugated with lipids have been used to create fluorescent sensors for pH, chloride, calcium, and oxygen that have been used to probe intracellular compartments and phagocytic pathways (a critical component of the immune response against infections) (Ji, et al. (2000), Anal Chem, 72:3497-503, Ji, et al. (2001), Anal Chem, 73:3521-7, Ma, et al. (2004), Anal Chem, 76:569-75, McNamara, et al. (2001), Anal Chem, 73:3240-6, Nguyen, et al. (2002), Anal Bioanal Chem, 374:69-74). However, the implementation of such probes has generally been limited to materials (usually synthetic dyes) that can be encapsulated within a lipid bilayer. Functional enzymes and membrane proteins have been much more difficult to capture within such structures (Walde, et al. (2001), Biomol Eng, 18:143-77). Moreover, synthetic lipid vesicles are difficult to localize and often lack the stability necessary for wide-spread application.
The specificity of membrane proteins can be controlled, in part, by engineering membrane proteins to bind antigen-specific antibodies. The antibody-binding (Z) domain of Staphylococcal Protein A (ProA) binds the constant, Fc, domain of IgG. Membrane-anchored antibodies and antibody-containing structures (ZZ-TM fusion proteins) have been incorporated into cells and viruses, primarily for use in targeting of gene therapy vectors (Bergman, et al. (2003), Virology, 316:337-47, Masood, et al. (2001), Int J Mol Med, 8:335-43, Morizono, et al. (2001), J Virol, 75:8016-20, Nakamura, et al. (2004), Nat Biotechnol, 22:331-6, Ohno, et al. (1997), Biochem Mol Med, 62:123-7, Ohno, et al. (1997), Nat Biotechnol, 15:763-7, Sawai, et al. (1998), Mol Genet Metab, 64:44-51, Snitkovsky, et al. (2000), J Virol, 74:9540-5). In all of the published cases, ZZ-TM fusion proteins bound antibody in the appropriate orientation for targeting of retroviruses to cells expressing complementary antigen.
The use of fluorescently-labeled beads as a solid substrate for binding reactions has recently become a popular replacement for traditional 96-well plates in such techniques as immunoassays and competitive binding assays. By flowing beads and their associated protein reactants through a kinetic fluidics system, and by incorporating into the beads a number of different dyes that can be detected after excitation at different wavelengths of light, sample throughput has been increased by allowing the simultaneous detection of multiple analytes (multiplex analysis).
Thus, there is a need for improvements in the performance of probes in the areas described above to allow superior spatial localization of abnormalities in vivo, as well as structure-function correlation on the subcellular level, and already have applications in the fields of cancer biology, cell biology, and gene therapy, for example in the detection of tumor markers and in tracking the delivery and function of gene therapy vectors. There is also a need for probes that can be spatially localized, that can amplify infrequent signals, and that can compartmentalize multiple reporters simultaneously. There is also a need to generate probes that can generate multimodal signals. The present invention satisfies the needs as well as others.
Ion channels are membrane-bound proteins which control the flow of ions across biological membranes, either through passive or active transport mechanisms. In the context of cell electrophysiology, ion channels are the primary molecular mechanism by which cells maintain a membrane potential. Membrane potential is generated and maintained by concentration gradients of charged ions such as sodium, potassium, chloride, hydrogen, and calcium, across the otherwise impermeable cell membrane. The membrane potential of a cell can change in the course of signaling, development, differentiation of function, and pathology.
Electrical potential differences are present across the plasma membrane of most living prokaryotic and eukaryotic cells, and also between the cytosol and the interior of organelles such as chloroplasts and mitochondria. As a consequence of ion concentration gradients that are maintained by active transport processes, the electrical membrane potential of some resting cells is approximately −70 mV, with the cell interior electrically negative with respect to the exterior. The membrane potential is reduced to zero when the plasma membrane is ruptured by chemical or physical agents. When a membrane is permeable to only a single ion species (the simplest theoretical model), the membrane potential is given by the Nernst equation: V=−(RT/zF)*ln([I]in/[I]out) where R is the gas constant, T is the absolute temperature, z is the ion valency, F is Faraday's constant, I is the cation concentration. The value of RT/F is 25.7 mV at 25° C.
A change in membrane potential in the positive direction is called a depolarization. Conversely, a change in membrane potential in the negative direction is called a hyperpolarization. Depolarization of the cell membrane during the action potential of a nerve or muscle cell typically results in the cell interior transiently becoming electrically positive with respect to the exterior, as Na-channels open, Na+ rushes in, and membrane potential approaches the Vion of Na+, +60 mV. Voltage-gated K-channels will open when there is sufficient depolarization, allowing K+ to rush out and bringing the membrane potential back to its resting value, approximately the Vion of K+.
Membrane potential is often measured by electrophysiological methods in which glass microelectrodes are inserted into or onto cells (e.g. voltage clamp, patch clamp) to directly measure the difference in electrical potential across the cell membrane. Many biological structures, however, are not readily amenable to microelectrode measurement, such as sub-cellular organelles and neuronal processes. Moreover, microelectrode techniques are difficult to automate for drug discovery applications.
In these cases, fluorescent dyes and probes that measure electrical membrane potential represent an alternative. Fluorescent dyes that can measure membrane potential were first employed in the 1970s, and have evolved in capability, speed of response, and sensitivity for the past thirty years (Cohen, et al. (1978), Rev Physiol Biochem Pharmacol, 83:35-88, Molecular Probes Handbook (2002), Plasek, et al. (1996), J of Photochemistry and Photobiology, 33:101-124, Smith (1990), Biochim Biophys Acta, 1016:1-28). Numerous studies have shown that optically and electrically recorded action potentials are identical in shape, and fluorescent dyes are now a well-accepted methodology for measuring electrical potential across a membrane. Since a single ion channel can facilitate the flux of a million ions per second, membrane potential change can be detected with high sensitivity even in cases of limited quantities of ion channel.
Many biological compartments, despite their importance in biology, are not readily amenable to ion or voltage measurements using conventional technologies. For example, some intracellular organelles are too small to seal with a microelectrode and cannot be readily labeled with a fluorescent dye or probe independent of the rest of the cell. Similarly, synaptic junctions are dynamic structures with budding vesicles, interstitial gaps, and secreted neurotransmitters and ions that cannot be easily isolated from the structure as a whole.
Thus, there remains a need for the development of an improved methodology that permits the study of ion channels and/or transporter proteins. The present invention satisfies this need. Further, there is a need for assays that permit the study of ion channel inhibitors and/or activators, and the present invention also satisfies this need as well others.
Viral vaccines are made in several ways, but all are significantly different than the lipoparticle. At least half a dozen different successful virus vaccine systems (summarized in Table 1) give support for using small particles to induce immune responses. Lipoparticles are unique because they are able to incorporate membrane proteins which are not part of the viral genome.
TABLE 1Killed virus vaccines (Field's Virology).Enveloped indicates whether virus is surrounded by a lipid bilayer.Virus VaccineEnvelopedType of VaccineHepatitis A−Whole inactivated virusHepatitis B+Recombinant virus-like particlesInfluenza A and B+Disrupted virusJapanese encephalitis virus+Whole inactivated virusPoliovirus−Whole inactivated virusRabies+Whole inactivated virus
Parvovirus-like particles (VLPs) and have been used to induce cytotoxic T lymphocyte (CTL) responses. This technology involves linking an antigen to the viral particle, which is significantly different than the lipoparticle which incorporates a membrane protein of interest into the lipid membrane of the cell. The Parvovirus VLP technology does not involve a lipid bilayer and therefore cannot allow a membrane protein to maintain its structure. Martinez et al (2003) used a recombinant VLP carrying a CD8(+) T cell determinant to successfully induce an immune response in mouse neonates (Martinez et al (2003) Virology. 2003; Jan. 20; 305(2):428-35). Wakabayashi et al (2002) fused the HPV16 E7 antigen to the major and minor capsid proteins (L1 and L2) of chimeric human papillomavirus (HPV) virus-like particles (cVLPs) (Wakabayashi et al Intervirology. 2002; 45(4-6):300-7). Mice vaccinated with this cVLP successfully generated a specific CTL response.
Antibodies are also useful in determining structure and function of a polypeptide, and can also be used as therapeutics. Polyclonal antibodies are a mixture of many antibodies recognizing different epitopes of an antigen. It is possible to isolate one of these antibodies and amplify it by fusing the antibody cell to an immortal tumor cell, forming a hybridoma. Amplification of the hybridoma results in a clonal population of cells that secrete antibodies which are identical; these antibodies recognize the same epitope on the same antigen, and are known as monoclonal antibodies (Mabs). Because of their specificity to a single epitope, Mabs that recognize non-linear epitopes (epitopes formed by a conformational structure of sequentially separated amino acids) are sensitive probes of subtle conformational changes brought about by either mutational alterations or variations in physical/chemical conditions.
Historically, it has been difficult to generate good Mabs to many integral membrane proteins. Traditional methods of immunization using purified proteins or peptides have limited application to membrane-bound receptors in which removal of the protein from a lipid environment results in complete loss of conformation. While antibodies can be elicited against peptides derived from extracellular sequences of topologically complex receptors, such antibodies often react with the native receptor inefficiently. The use of whole cells has been used with success for the development of some antibodies to complex receptors (Lee, et al. (1999), J. Biol. Chem., 274:9617-9626), but has a number of limitations. For example, many receptors, especially when over expressed within a cell, can reduce cell health, cause cell death, be cycled away from the cell surface, aggregate, denature, or have difficulty being translated, leading to poor expression. In addition, the protein of interest is typically a minor component on the cell surface, and the numbers of Mabs elicited by this approach is typically small.
The chemokine receptors CCR5 and CXCR4 serve as examples of integral membrane proteins which are difficult to generate Mabs against. CCR5 and CXCR4 are GPCRs that in addition to their normal functions in the immune system are also used by HIV to infect cells. Due to their importance for virus infection, considerable effort has been spent on developing immunological reagents to these and related receptors. Some of the first CCR5 and CXCR4 receptor antibodies were obtained via immunization with peptides (Doranz, et al. (1997), J. Virol., 71:6305-6314). However, second-generation antibodies obtained following immunization of mice with cells over-expressing the receptor of interest supplanted these first generation antibodies (Lee, et al. (1999), J. Biol. Chem., 274:9617-9626). While such an approach is labor intensive and only a very small fraction of hybridomas target the desired receptor, it is now clear that presentation of GPCRs in their native conformation is essential for the generation of effective Mabs. The solved structure of bovine rhodopsin, a 7 transmembrane (7TM) receptor, as well as structure-function studies on a variety of 7TM receptors including CCR5 and CXCR4, indicate that the extracellular domains of these proteins are conformationally complex. In addition, disulfide bonds link the amino terminal domain of these receptors with the third extracellular loop, and the first with the second extracellular loops. Thus, it is not surprising that the epitopes recognized by many Mabs are composed of residues from multiple extracellular domains of the receptor. Therefore, the native conformation of the receptor as it resides in the membrane can be of critical importance not just for its function, but also for the elicitation of specific, high affinity Mabs.
The antigenic structures of topologically complex proteins such as GPCRs, amino acid transporters, and ion channels are critically influenced by the lipid environment; a large fraction of the protein's mass, sometimes even a majority, is embedded in the lipid bilayer. Attempts to reconstitute such receptors in artificial membranes have proven difficult, though not impossible. In our experience, the most useful Mabs to 7TM proteins have thus far come from using cells for immunization that express the receptors in their native conformation. While this approach has proven successful for the generation of Mabs to the chemokine receptors CXCR4, APJ, CCR2, CCR3, and CCR5, it has proven difficult for the generation of Mabs to the chemokine receptors XCR1 (the Lymphotactin receptor), CCR4, CCR7, CCR8, and CX3CR1 (the Fractalkine receptor). In addition, most attempts have failed to produce high affinity Mabs to topologically complex receptors such as glucose transporters (e.g. Glut4), ion channels (e.g. Kv1.3), and amino acid transporters (e.g. MCAT1).
Thus, there is a need for compositions that facilitate the elicitation of an immune response against native membrane protein structures, particularly for the generation of monoclonal antibodies, of humoral response, of cellular response, and for vaccines, and the present invention also satisfies this need. The present invention fulfills these needs as well others.
Protein transfection is defined as the internalization of an exogenous protein into a target cell's cytoplasm or nucleus. While similar to DNA transfection in that both processes ultimately introduce exogenous protein into a cell, protein transfection differs by bypassing the transcription and translation machinery needed for protein production from DNA. Furthermore, by introducing a foreign protein directly into the target cell, protein transfection introduces the protein faster than DNA transfection and bypasses potential complications of DNA transfection and protein synthesis, such as undesired or incorrect RNA splicing variations, protein folding, and post-translational modifications.
Current applications for protein transfection include studying proteins that are inherently toxic to cells, that are involved in signaling cascades within cells, and that are downstream cascade regulators. For example, antibodies transfected into the cytoplasm can be used to inhibit or modify downstream signaling cascade effectors (Marrero 1995; Rui 2002). Additionally, fluorescent ligands and substrates can be transfected into a target cell's cytoplasm or nucleus to study a signaling pathway of interest (Nolkrantz 2002).
Previous protein transfection techniques include microinjection, myristylation of amino termini, protein encapsulation by various lipid formulations, such as those comprising cationic lipids, and the use of peptides that bind to the protein that is being transfected (e.g. Protein Transduction Domains (PTDs). Current commercial kits include Pro-Ject (Pierce Biotechnology, Inc.) and Profect (Targeting Systems) both using lipid-based technologies, and Chariot™, protein delivery reagent, (Active Motif) using PTD technology. Other methods of protein transfection include the HIV Tat protein. However, current methods still do not resolve the problems of transfecting membrane proteins into a cell. Most, if not all, membrane proteins require proper folding and structure to be active. The current methods of transfecting membrane proteins are inadequate and can be improved.
Thus, there is a need for new and improved protein transfection methods and compositions to introduce proteins into cells. The present invention fulfills this need as well as others.
Viruses are of great significance to the field of medicine, but there are few techniques for quantifying and characterizing viruses due to their exceptionally small size. Most viruses are between 30 nm and 1 μm, too small for direct visualization under a light microscope or with the use of most cellular detection methods. Quantification of viruses can be especially difficult, with most researchers relying on infectious assays for quantification, an assay that typically takes several days, requires live virus, and detects only infectious virions. Many infectious viruses are produced along with 10 to 100-fold more non-infectious viruses, and these non-infectious viruses are rarely quantified (Knipe, et al. (2001)). Visualization of fluorescent viruses has been used in some cases to overcome these obstacles (Leopold, et al. (2000), Hum Gene Ther, 11:151-65, McDonald, et al. (2002), J. Cell Biol., 159: McDonald, et al. (2003), Science, 300:1295-7, Seisenberger, et al. (2001), Science, 294:1929-32).
In addition, the proteins that reside on the surface of lipid-enveloped viruses are also difficult to detect and quantify. Previous research has disclosed methods of detecting some membrane proteins on the surface of intact HIV and SIV virions (Bastiani, et al. (1997), J. Virol., 71:3444-3450, Capobianchi, et al. (1994), J Infect Dis, 169:886-9, Nyambi, et al. (2001), J Immunol Methods, 253:253-62, Orentas, et al. (1993), AIDS Res Human Retroviruses, 9:1157-1165). In addition, research has demonstrated the detection of membrane proteins on a virus-like particle using confocal microscopy (Zemanova, et al. (2004), Biochemistry). However, many membrane proteins can be relatively sparse, difficult to detect, and even more difficult to quantify on an absolute basis (Coorssen, et al. (2002), Anal Biochem, 307:54-62). Yet such membrane proteins, such as the native viral envelope protein on HIV, gp160, can be central to the infectious life cycle of the virus and the pathogenesis of the disease it causes.
Furthermore, membrane proteins compose a complex structure that is often dependent on the lipid bilayer in which they reside. Methods to measure the structural state of the membrane protein within an enveloped virus are needed. When the protein of interest is Envelope, infection assays have been used, but in cases where the membrane protein does not mediate viral entry other methods are needed.
Currently, the most commonly employed assays for protein interactions include radioimmunoassays, competitive protein binding assays, and enzyme-linked immunoassays. All of these, however, suffer from a number of drawbacks: they are expensive, labor-intensive, time-consuming, and have not been used to study membrane proteins without the use of whole cells or membrane vesicles derived from cells. One of the major limitations to many in vitro analyses of protein binding is the requirement for the protein(s) of interest to be structurally intact and in solution in order for appropriate interaction with receptor to occur. Many proteins of interest, including the receptors of many viral envelope proteins, are integrated into cell membranes. These proteins are notoriously difficult to purify and solubilize in their structurally-intact form, limiting the ability to work with them. As such, laboratory techniques involving such proteins are either non-existent or involve laborious preparatory steps.
Thus, there remains a need for new and/or improved methods for detecting and quantifying viruses, viral particles, virus like particles and lipoparticles. There is also a need for new and/or improved methods and techniques to detect and quantify the membrane proteins that reside in viruses, viral particles, and lipoparticles. There is also a need for improved methods of identifying proteins, antibodies, ligands, or drugs that bind to these membrane proteins. The present invention satisfies these needs and others.
Sensors capable of detecting infectious agents and/or of detecting a serological reaction in people exposed to dangerous pathogens, are desired for rapid detection and diagnosis of infectious disease, for screening for environmental and food contaminants, and for efficient biodefense screening and response procedures (reviewed in (Iqbal, et al. (2000), Biosens Bioelectron, 15:549-78)). Flaviviruses, for example, are a group of positive-stranded RNA viruses that have a global impact resulting from their widespread distribution and ability to cause disease in humans and economically important domestic animals. Several members of this genus, such as dengue virus (DEN) and West Nile virus (WNV), are considered emerging or re-emerging pathogens because of the rapid annual increase in the rate at which they encounter humans and cause disease (Gubler (1998), Clin Microbiol Rev, 11:480-96). With 50 million cases of related illness reported annually, DEN infection has become the most significant source of arthropod-borne viral disease in humans (Gubler, et al. (1993), Infect Agents Dis, 2:383-93, Monath (1994), Proc Natl Acad Sci USA, 91:2395-400). Both DEN and WNV are emerging biodefense pathogens (category A and B, respectively) for which the development of diagnostics, therapeutics, and vaccines are the focus of considerable effort. The development of a DEN vaccine is particularly challenging because sequential exposure to different serotypes of DEN actually increases (rather than attenuates) the likelihood of developing dengue hemorrhagic fever in response to infection (Burke, et al. (1988), Am J Trop Med Hyg, 38:172-80, Graham, et al. (1999), Am J Trop Med Hyg, 61:412-9, Sangkawibha, et al. (1984), Am J Epidemiol, 120:653-69, Vaughn, et al. (2000), J Infect Dis, 181:2-9, Winter, et al. (1968), Am J Trop Med Hyg, 17:590-599). For such pathogens, characterizing not only the magnitude, but also the breadth, persistence, and specificity of the humoral response is an important component of evaluating candidate vaccines and understanding pathogenesis in infected individuals.
Conventional laboratory techniques used to detect pathogens and antibodies in blood serum and other samples include ELISA, PCR, and cell culture (Belgrader, et al. (1999), Science, 284:449-50, Belgrader, et al. (2003), Anal Chem, 75:3114-8, Rowe, et al. (1999), Anal Chem, 71:3846-52). A common limitation of these techniques is the time and complexity of the assays themselves, which usually require extensive sample handling. This renders them unsuitable for rapid screening of samples, for automation, and for portability for field applications. Recent directions in the development of alternative techniques for pathogen detection reflect these limitations in traditional technology, and the need for increased simplicity, reliability, and rapidity for methods that detect both biodefense pathogens and antibodies that target them.
Increasingly, biosensor systems are utilizing living cells, and their complex sensing and signaling mechanisms, for assays such as pathogen detection (Bechor, et al. (2002), J Biotechnol, 94:125-32, Belkin (2003), Curr Opin Microbiol, 6:206-12, Conway, et al. (2002), Receptors Channels, 8:331-41, Haruyama (2003), Adv Drug Deliv Rev, 55:393-401, Kamei, et al. (2003), Biotechnol Lett, 25:321-5, Karube, et al. (1994), Curr Opin Biotechnol, 5:54-9, Park, et al. (2003), Biotechnol Prog, 19:243-53). Cell-based biosensors are already being exploited in diagnostic and screening tests, including some for biodefense pathogens. For example, a technique was reported (Rider, et al. (2003), Science, 301:213-5) in which B-cells were used to report the presence of clone-specific pathogens. These cells were transfected with a calcium-sensitive bioluminescent protein that was sensitive to fluctuations in cytosolic calcium resulting from B-cell receptor (BCR) signaling. Cell-based assays of this sort show improvements in fidelity, simplicity, and speed when compared with traditional pathogen detection techniques. However, despite their advantages compared with traditional methods, assays that utilize living cells are limited in three main areas: 1) Dependence on living cells that require high maintenance and specialized tissue culture facilities, rendering miniaturization (due to cell size and environmental requirements) and field application impractical or impossible. 2) Limitation in flexibility of antigen recognition due to the clonal nature of cell pathogen-receptors (such as BCRs), requiring extensive cell line development for detection of diverse pathogens or of mutants and variants. 3) Susceptibility to false positives resulting from reliance on a single pathogen receptor, and on detection of a single downstream (and often promiscuous) signaling event (e.g. Ca++ flux). There is a need for the development of pathogen (and ligand) biosensors that can utilize cell-sensing and signaling machinery in a flexible and cell-free format in order to overcome these limitations.
Sensors that take advantage of the signaling capacity of single-transmembrane (1-TM) proteins, such as BCRs and kinase-activating receptors, have not been utilized outside of laboratory-based live-cell assays. Cells have been required for both the maintenance of the native structure of 1-TM proteins (which is reliant upon the presence of the cell membrane), and for the retention of cellular signaling pathways that can be linked to a measurable reporter. 1-TM receptors typically comprise an extracellular ligand recognition domain, a transmembrane domain that crosses the cell membrane once and anchors the protein to the cell surface, and one or more intracellular domains which interact directly or indirectly with cytosolic signaling proteins. Functionally, many 1-TM receptors mediate their signaling activities through a common mechanism—ligand-induced receptor cross-linking. For example, BCRs exist as unliganded monomers on the cell surface that, when cross-linked by an appropriate antigen, form clusters on the cell surface. Clustering and cross-linking of 1-TM receptors induces a cascade of phosphorylation events that recruits adaptors and other accessory proteins to the receptor complex, ultimately activating multiple signaling pathways such as calcium/calmodulin, phospholipase C, Ras, and MAPK. The ability to easily manipulate 1-TM pathogen and ligand recognition elements, and link them to signaling and reporter (output) pathways in cell-free vehicles, could enable new types of pathogen sensors to be developed, exhibiting many of the advantages of live-cell assays, but without their practical limitations.
One method of measuring protein-protein interactions is by fluorescent resonance energy transfer (FRET). When complementary fluorescent reporters are brought into close proximity, the transfer of fluorescent energy from an excited donor (CFP) to an acceptor (YFP) results in fluorescence emission by the acceptor (Stanley (2003), Chroma Application Note No. 6). The transfer of energy is by non-radiative dipole-dipole interaction, making FRET efficiency highly dependent upon fluorochrome pair proximity (within 5 nm), and thus an excellent indicator of proximity. FRET strategies have been used on numerous occasions within cells and cell-based biosensors to measure interactions between membrane proteins (Chan, et al. (2001), Cytometry, 44:361-8, Minor (2003), Curr Opin Drug Discov Devel, 6:760-5, Overton, et al. (2000), Curr Biol, 10:341-4, Overton, et al. (2002), Methods, 27:324-32, Tertoolen, et al. (2001), BMC Cell Biol, 2:8). A similar system using a luminescent and fluorescent pair is also available (BRET).
Thus there is a need for improved molecules and methods for the detection of antigens and ligands. The present invention fulfils these needs as well as others.
G protein coupled receptors (GPCRs) are a large family of cell surface receptors with an assortment of ligands and diverse biological actions. The importance of GPCRs in cellular function, their diversity, and their accessibility to exogenous agents make them an important focus of research into disease processes and drug discovery.
GPCR activation events are communicated to cell signaling pathways via GTP-binding proteins (G proteins) associated with the intracellular domain of the receptor. GPCRs constitute the largest group of drug targets today, highlighting their importance in biological research and in disease pathways. However, GPCRs are structurally complex, spanning the cell membrane seven times. Removal from the cell membrane usually destroys the receptor's native structure which is maintained by the environment of the lipid bilayer. GPCRs are thus extremely difficult to purify and manipulate experimentally, and their study relies on whole cells or isolated cell membranes. However, these formats suffer from poor receptor purity, stringent environmental requirements, and an inability to be miniaturized, prohibiting their application to emerging micro- and nano-scale detection technologies. Methods for assaying GPCR activation that can be applied to microfluidic drug-screening devices are needed.
Although GPCRs respond to a wide variety of extracellular ligands, they mediate intracellular communication through common signaling pathways (Kiselyov, et al. (2003), Cell Signal, 15:243-53, Morris, et al. (1999), Physiol Rev, 79:1373-430). The intracellular domains of GPCRs are coupled to a heterotrimeric complex of membrane-associated GTP-binding proteins (G proteins). Nearly all GPCRs initiate their signaling pathway through the action of G proteins, which transmit the GPCR activation signal to intracellular effectors. The G protein complex consists of Gα, Gβ, and Gγ subunits, each of which occur as a number of ligand- and signal-specific isotypes. For example, the Go family includes Gi, Gs, Gq, and G12 isotypes, and a family such as Gi is composed of several sub-members (Gi, Go, Gt, Ggus, and Gz). In the inactive state, the Gα subunit binds GDP and maintains the GPCR in a ligand-receptive conformation. GPCR stimulation by an agonist induces Gα to exchange GDP for GTP. The now activated G protein subunits dissociate and activate signaling cascades that release second messengers such as cAMP and intracellular calcium. These second messengers exert their biological effects by modifying cellular processes such as gene expression, ion balance, and the release of bioactive substances. The hydrolysis of GTP to GDP by Gα returns the G proteins to their inactive state, attenuating and eventually terminating the signal. Multiple accessory proteins, such as arrestins and GTPase-activating proteins (GAPs), modify these downstream signaling events. A number of studies have created GPCR-G protein fusion proteins (Milligan (2000), Trends Pharmacol Sci, 21:24-8, Milligan (2002), Method in Enzymology: G Protein Pathways Part A, 343:260-273, Molinari, et al. (2003), J Biol Chem, 278:15778-88).
GPCR activation is traditionally measured experimentally by monitoring one or more of the participants of these signaling cascades. Fluorescently or radioactively labeled GPCRs, G-proteins, and guanine nucleotides, have all been cited as potential reporters of intracellular signaling events (Eidne, et al. (2002), Trends Endocrinol Metab, 13:415-21, Hemmila, et al. (2002), Drug Discov Today, 7:S150-6, Kimple, et al. (2001), J Biol Chem, 276:29275-81, Milligan (2003), Trends Pharmacol Sci, 24:87-90, Moore, et al. (1993), Biochemistry, 32:7451-9, Remmers (1998), Anal Biochem, 257:89-94, Remmers, et al. (1996), J Biol Chem, 271:4791-7, Remmers, et al. (1994), J Biol Chem, 269:13771-8). However, ‘end-point’ messengers such as calcium flux or cAMP production are not stimulated by a number of important GPCRs and G proteins, and as such, the ligands and functions of many GPCRs remain unknown.
Thus there is a need for improved methods for detecting GPCR activation to overcome the current constraints by being simple, flexible, and applicable to a wide range of GPCRs and G-proteins. The present invention fulfils these needs as well as others.
Proteins that span the membrane multiple times present a unique set of challenges for structural analyses such as x-ray crystallography. As a result, the structure of only a handful of multiple-spanning membrane proteins has been determined at high resolution (for example, see (Jiang, et al. (2002), Nature, 417:515-22, Jiang, et al. (2003), Nature, 423:33-41, Palczewski, et al. (2000), Science, 289:739-45, Pebay-Peyroula, et al. (1997), Science, 277:1676-1681) (approximately 40 unique membrane protein structures compared to 3,000 unique soluble protein structures) (Nollert, et al. (2004), DDT: Targets, 3:2-4, Werten, et al. (2002), FEBS Lett, 529:65-72), and the vast majority of these proteins are relatively simple, prokaryotic proteins (52 of 67 total membrane protein structures are of bacterial origin) (Werten, et al. (2002), FEBS Lett, 529:65-72).
Structural studies of any protein typically require the protein to be expressed at high levels, solubilized for manipulation, and purified to near complete homogeneity. Integral membrane proteins present difficulties in all three of these requirements. While soluble (secreted or cytoplasmic) proteins can be expressed at high levels within traditional E. coli and insect cell expression systems, membrane proteins have been less successful, in part because these systems lack many of the (poorly understood) folding, membrane insertion, and trafficking mechanisms used to express higher-order eukaryotic membrane proteins. Oligomeric membrane proteins present even greater difficulties, as co-expression of subunits and assembly systems are required for efficient production of structurally intact functional units. In addition, the membrane surface area of cells is limited, restricting the quantity of membrane protein that can be expressed in any given cell (e.g. compared to secreted and intracellular proteins). Exemplifying this limitation, rhodopsin, the sole GPCR for which the crystal structure has been determined, was derived from natural tissue (bovine retinas) that contain unusually large amounts of the protein (Palczewski, et al. (2000), Science, 289:739-45). Comparable natural sources for other membrane proteins are rarely available. Despite these difficulties, however, several membrane proteins have been successfully expressed at high levels, often using baculovirus insect cell expression systems (Klaassen, et al. (1999), Biochem J, 342 (Pt 2):293-300, Lundstrom (2003), Biochim Biophys Acta, 1610:90-6, Massotte (2003), Biochim Biophys Acta, 1610:77-89, Nollert, et al. (2004), DDT: Targets, 3:2-4). Mammalian expression systems, such as semliki forest virus (SFV), have also been used to obtain large quantities of membrane proteins (Lundstrom (1997), Curr Opin Biotechnol, 8:578-82, Lundstrom (2003), Biochim Biophys Acta, 1610:90-6, Lundstrom, et al. (2001), FEBS Lett, 504:99-103, Wurm, et al. (1999), Curr Opin Biotechnol, 10:156-9). Just as importantly, advances in crystallization have extended the useful life of limited quantities of protein by miniaturizing crystallization trials (2 mg of protein is often now sufficient for an entire trial).
Despite the successful large-scale expression of at least some membrane proteins, membrane proteins are still difficult to crystallize. A primary reason for this disparity is because once expressed, membrane proteins face another obstacle—purification to homogeneity. While soluble proteins can be readily purified from the cellular media (in the case of secreted proteins) or soluble cell fractions (in the case of cytoplasmic proteins), membrane proteins remain embedded within the cell where they are difficult to extract. A significant portion of many membrane protein molecules, sometimes even a majority of the protein, is embedded within the plasma membrane lipid bilayer. GPCRs for example, possess seven distinct transmembrane domains, making large portions of these proteins hydrophobic and the protein as a whole topologically complex. Removal of GPCRs (and most other multi-spanning membrane proteins) from their lipid bilayer usually results in loss of their native structure. Because 95% of the cell's membrane content is inside the cell (nucleus, mitochondria, endoplasmic reticulum, golgi, etc.), purification of plasma membrane proteins is not trivial. Detergents can allow solubilization of some membrane proteins in their native state, but this is an empirical and very time-consuming process, and even then stability is only transient. Finding a detergent that keeps a membrane protein structurally intact, in micelle form, and stable enough for purification is difficult; finding one that is selective enough to accomplish these things in the presence of a large amount of contaminating lipid and protein limits the choices of detergent even further. Finally, even when membrane proteins can be expressed and purified from cells using detergents, cell lysates will contain a heterogeneous mix of the membrane protein of interest in various stages of synthesis, folding, and processing.
Therefore, there is a need for innovations that focus primarily on the development of methods for membrane protein preparation. A novel method that can enable the purification of large quantities of homogeneous membrane protein could have a major impact on drug discovery and membrane protein research.
Baculovirus vectors are commonly used to express high quantities of membrane proteins in their correctly folded, natively processed forms (Carfi, et al. (2002), Acta Crystallogr D Biol Crystallogr, 58:836-8, Carfi, et al. (2001), Mol Cell, 8:169-79, Klaassen, et al. (1999), Biochem J, 342 (Pt 2):293-300, Massotte (2003), Biochim Biophys Acta, 1610:77-89). Baculovirus systems are limited to particular cell types (most commonly insect Sf9 or High Five cells), but result in very high levels of expression from the polyhedrin promoter in serum-free growth conditions. Insect cell-derived proteins are routinely used in crystallography studies, a result of their combined high protein yield and ability to produce correctly folded and processed eukaryotic proteins. Baculovirus vectors expressing Gag (from HIV and from MLV retroviruses) have previously been used to study retroviral assembly mechanisms (Adamson, et al. (2003), Virology, 314:488-96, Gheysen, et al. (1989), Cell, 59:103-12, Hughes, et al. (1993), Virology, 193:242-55, Royer, et al. (1992), J Virol, 66:3230-5, Yamshchikov, et al. (1995), Virology, 214:50-8, Yao, et al. (2003), Vaccine, 21:638-43, Yao, et al. (2000), AIDS Res Hum Retroviruses, 16:227-36, Zemanova, et al. (2004), Biochemistry).
Vaccinia virus vectors are one of the most potent expression systems within mammalian cells. Vaccinia is capable of infecting nearly any cell type and expresses high amounts of protein driven from an internal vaccinia promoter (synthetic early-late promoter). Both transcription and translation of vaccinia genes occur in the infected cell's cytoplasm, enabling high level expression of nearly any protein. Vaccinia expressing Gag proteins have previously been described as a means of studying retroviral assembly and budding (Karacostas, et al. (1989), Proc Natl Acad Sci USA, 86:8964-7). Vaccinia vectors can be easily produced by recombination between a specially engineered plasmid (psC60) and a wild type strain of the virus (WR), followed by plaque purification. Advantages of vaccinia also include native mammalian cell processing and trafficking. Replication-deficient vaccinia virus MVA can also be used to reduce virus-induced toxicity and the presence of live virus.
Alphaviruses, such as semliki forest virus (SFV), have proven to be among the most robust mammalian expression systems described to date, especially for correctly folded and processed eukaryotic membrane proteins (Lundstrom (1997), Curr Opin Biotechnol, 8:578-82, Lundstrom (2003), Biochim Biophys Acta, 1610:90-6, Lundstrom, et al. (2001), FEBS Lett, 504:99-103, Wurm, et al. (1999), Curr Opin Biotechnol, 10:156-9). Their utility reflects three major attributes. First, the genetic organization of the SFV genome allows the introduction of heterologous genes in place of genes encoding the viral structural proteins, where they are under the control of an internal sub-genomic promoter (26S). Second, RNAs encoded by alphavirus vectors (called replicons) are capable of cytoplasmic replication in transduced cells. Replication of the replicon RNA in the cytoplasm effectively increases the number of templates for transcription and bypasses mRNA nuclear export limitations, resulting in high-level gene expression. Finally, when SFV structural genes are provided in trans, SFV replicons can be packaged into virus particles capable of single-round infection of virtually any cell type. SFV vectors expressing Gag (from HIV and from MLV retroviruses) have previously been used to study viral assembly and to produce more effective vaccines (L1, et al. (1996), Proc Natl Acad Sci USA, 93:11658-63, Suomalainen, et al. (1994), J Virol, 68:4879-89, Weclewicz, et al. (1998), J Virol, 72:2832-45). While SFV is capable of high levels of protein expression within hours of infection, the virus normally kills infected cells within several days. Mutations of SFV have been characterized that delay or prevent cytotoxicity that can be used (Lundstrom, et al. (2001), FEBS Lett, 504:99-103). Numerous alternative alphavirus expression systems exist (pSFV-help, Invitrogen) and/or are emerging (producer cells with capsid-E1-E2/3) that continue to improved ease of use and viral titer.
Recombinant adenovirus expressing Gag has previously been developed as a means of producing retroviruses for gene therapy (Caplen, et al. (1999), Gene Ther, 6:454-9, Duisit, et al. (1999), Human Gene Therapy, 10:189-2000, Lin (1998), Gene Therapy, 9:1251-1258, Ramsey, et al. (1998), Biochem Biophys Res Commun, 246:912-9, Torrent, et al. (2000), Cancer Gene Ther, 7:1135-44, Yoshida, et al. (1997), Biochem Biophys Res Commun, 232:379-82).